Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica.

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.

A Journey from Structure to Function of Bacterial Lipopolysaccharides.

Lipopolysaccharide (LPS) is a crucial constituent of the outer membrane of most Gram-negative bacteria, playing a fundamental role in the protection of bacteria from environmental stress factors, in drug resistance, in pathogenesis, and in symbiosis. During the last decades, LPS has been thoroughly dissected, and massive information on this fascinating biomolecule is now available. In this Review, we will give the reader a third millennium update of the current knowledge of LPS with key information on the inherent peculiar carbohydrate chemistry due to often puzzling sugar residues that are uniquely found on it. Then, we will drive the reader through the complex and multifarious immunological outcomes that any given LPS can raise, which is strictly dependent on its chemical structure. Further, we will argue about issues that still remain unresolved and that would represent the immediate future of LPS research. It is critical to address these points to complete our notions on LPS chemistry, functions, and roles, in turn leading to innovative ways to manipulate the processes involving such a still controversial and intriguing biomolecule.

Biophysical and Structural Characterization of the Interaction between Human Galectin-3 and the Lipopolysaccharide from Pseudomonas aeruginosa.

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

Synthesis of Unprecedented α/ß-Alternate (1→4)-Glucans via Stereoselective Iterative Glycosylation.

Glucans are major biomaterials on the earth, with α-(1→4)-glucans (i. e., amylose) and ß-(1→4)-glucans (i. e., cellulose) being the most abundant ones, which are relevant to energy storage and structural function, respectively. Interestingly, (1→4)-glucans with alternate α/ß-linkages, namely herewith amycellulose, have never been disclosed in nature. Here we report a robust glycosylation protocol for the stereoselective construction of the 1,2-cis-α- and 1,2-trans-ß-glucosidic linkages, which employs an optimal combination of glycosyl N-phenyltrifluoroacetimidates as donors, TMSNTf2 as promoter, CH2 Cl2 /nitrile or CH2 Cl2 /THF as solvents. A broad substrate scope has been demonstrated by coupling five imidate donors with eight glycosyl acceptors, in which most of the glycosylations lead to high yield and exclusively 1,2-cis-α- or 1,2-trans-ß-selectivity. Applying this glycosylation protocol and with an iterative manner, the unprecedented α/ß-alternate (1→4)-glucans up to a 16-mer have been synthesized. Differently from amylose, that adopts a compact helicoidal arrangement, the synthetic amycellulose features an extended ribbon-like conformation, comparable to the extended shape of cellulose.

Promoter-Controlled Synthesis and Conformational Analysis of Cyclic Mannosides up to a 32-mer.

Cyclodextrins are widely used as carriers of small molecules for drug delivery owing to their remarkable host properties and excellent biocompatibility. However, cyclic oligosaccharides with different sizes and shapes are limited. Cycloglycosylation of ultra-large bifunctional saccharide precursors is challenging due to the constrained conformational spaces. Herein we report a promoter-controlled cycloglycosylation approach for the synthesis of cyclic α-(1→6)-linked mannosides up to a 32-mer. Cycloglycosylation of the bifunctional thioglycosides and (Z)-ynenoates was found to be highly dependent on the promoters. In particular, a sufficient amount of a gold(I) complex played a key role in the proper preorganization of the ultra-large cyclic transition state, providing a cyclic 32-mer polymannoside, which represents the largest synthetic cyclic polysaccharide to date. NMR experiments and a computational study revealed that the cyclic 2-mer, 4-mer, 8-mer, 16-mer, and 32-mer mannosides adopted different conformational states and shapes.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

Structural determination of the lipid A from the deep-sea bacterium Zunongwangia profunda SM-A87: a small-scale approach.

Zunongwangia profunda SM-A87 is a deep-sea sedimentary bacterium from the phylum Bacteroidetes, representing a new genus of Flavobacteriaceae. It was previously investigated for its capability of yielding high quantities of capsular polysaccharides (CPS) with interesting rheological properties, including high viscosity and tolerance to high salinities and temperatures. However, as a Gram-negative, Z. profunda SM-A87 also expresses lipopolysaccharides (LPS) as the main components of the external leaflet of its outer membrane. Here, we describe the isolation and characterization of the glycolipid part of this LPS, i.e. the lipid A, which was achieved by-passing the extraction procedure of the full LPS and by working on the ethanol precipitation product, which contained both the CPS fraction and bacterial cells. To this aim a dual approach was adopted and all analyses confirmed the isolation of Z. profunda SM-A87 lipid A that turned out to be a blend of species with high levels of heterogeneity both in the acylation and phosphorylation pattern, as well as in the hydrophilic backbone composition. Mono-phosphorylated tetra- and penta-acylated lipid A species were identified and characterized by a high content of branched, odd-numbered, and unsaturated fatty acid chains as well as, for some species, by the presence of a hybrid disaccharide backbone.

Exploring the Molecular Interactions of Symmetrical and Unsymmetrical Selenoglycosides with Human Galectin-1 and Galectin-3.

Galectins (Gals) are small cytosolic proteins that bind ß-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

Total Synthesis and Stereochemistry Assignment of Nucleoside Antibiotic A-94964.

A collective total synthesis of eight diastereoisomers associated with NMR analysis leads to a full stereochemistry assignment of the structurally unique nucleoside antibiotic A-94964, which features an octuronic acid uridine core decorated with an α-D-mannopyranosyl residue and an α-D-N-acylglucosaminopyranosyl residue via a phosphodiester bridge.

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis.

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing ß-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

Analysis of Synthetic Monodisperse Polysaccharides by Wide Mass Range Ultrahigh-Resolution MALDI Mass Spectrometry.

Carbohydrates, such as oligo- and polysaccharides, are highly abundant biopolymers that are involved in numerous processes. The study of their structure and functions is commonly based on a material that is isolated from complex natural sources. However, a more precise analysis requires pure compounds with well-defined structures that can be obtained from chemical or enzymatic syntheses. Novel synthetic strategies have increased the accessibility of larger monodisperse polysaccharides, posing a challenge to the analytical methods used for their molecular characterization. Here, we present wide mass range ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) as a powerful platform for the analysis of synthetic oligo- and polysaccharides. Synthetic carbohydrates 16-, 64-, 100-, and 151-mers were mass analyzed and characterized by MALDI in-source decay FT-ICR MS. Detection of fragment ions generated from glycosidic bond cleavage (or cross-ring cleavage) provided information of the monosaccharide content and the linkage type, allowing for the corroboration of the carbohydrate compositions and structures.

The Peculiar Structure of Acetobacter pasteurianus CIP103108 LPS Core Oligosaccharide.

Acetobacter pasteurianus, a member of the Alphaproteobacteria, is an acetic acid-producing bacterium present on sugar-rich substrates such as such as fruits, flowers and vegetables and traditionally used in the production of fermented food. The preferred living habitat associated with acid conditions makes the structure of the bacterial cell wall interesting to study, due to expected uncommon features. We have used a combination of chemical, analytical and NMR spectroscopy approaches to define the complete structure of the core oligosaccharide from A. pasteurianus CIP103108 LPS. Interestingly, the core oligosaccharide displays a high concentration of negatively charged groups, structural features that might contribute to reinforcing the bacterial membrane.

A chronic strain of the cystic fibrosis pathogen Pandoraea pulmonicola expresses a heterogenous hypo-acylated lipid A.

Pandoraea sp. is an emerging Gram-negative pathogen in cystic fibrosis causing severe and persistent inflammation and damage of the lungs. The molecular mechanisms underlying the high pathogenicity of Pandoraea species are still largely unknown. As Gram-negatives, Pandoraea sp. express lipopolysaccharides (LPS) whose recognition by the host immune system triggers an inflammatory response aimed at the bacterial eradication from the infected tissues. The degree of the inflammatory response strongly relies on the fine structure of the LPS and, in particular, of its glycolipid moiety, i.e. the lipid A. Here we report the structure of the lipid A isolated from the LPS of a chronic strain of P. pulmonicola (RL 8228), one of the most virulent identified so far among the Pandoraea species. Our data demonstrated that the examined chronic strain produces a smooth-type LPS with a complex mixture of hypoacylated lipid A species displaying, among other uncommon characteristics, the 2-hydroxylation of some of the acyl chains and the substitution by an additional glucosamine on one or both the phosphate groups.

Semisynthetic Isomers of Fucosylated Chondroitin Sulfate Polysaccharides with Fucosyl Branches at a Non-Natural Site.

The several interesting activities detected for fucosylated chondroitin sulfate (fCS) have fueled in the last years several efforts toward the obtainment of fCS oligosaccharides and low molecular weight (LMW) polysaccharides with a well-defined structure, in order to avoid the problems associated with the potential employment of native, sea cucumber sourced fCSs as a drug. Total synthesis and controlled depolymerization of the natural fCS polysaccharides are the main approaches to this aim; nonetheless, they present some limitations. These could be circumvented by semisynthesis, a strategy relying upon the regioselective fucosylation and sulfation of a microbial sourced polysaccharide sharing the same chondroitin backbone of fCS but devoid of any fucose (Fuc) and sulfate decoration on it. This approach is highly versatile, as it could open access also to fCS isomers carrying Fuc and sulfate groups at non-natural sites. Here we prepare for the first time some structurally homogeneous fCS isomers through a multistep procedure with a glycosylation reaction between a LMW polysaccharide acceptor and three different Fuc donors as key step. The obtained products were subjected to a detailed structural characterization by 2D-NMR. The conformational behavior was also investigated by NMR and molecular dynamics simulation methods and compared with data reported for natural fCS.

Chemical Synthesis of Sialyl N-Glycans and Analysis of Their Recognition by Neuraminidase.

The chemical synthesis of a fully sialylated tetraantennary N-glycan has been achieved for the first time by using the diacetyl strategy, in which NHAc is protected as NAc2 to improve reactivity by preventing intermolecular hydrogen bonds. Another key was the glycosylation to the branched mannose in an ether solvent, which promoted the desired glycosylation by stabilizing the oxocarbenium ion intermediate. Furthermore, high α-selectivity of these glycosylation reactions was realized by utilizing remote participation. Two asymmetrically deuterium labeled sialyl N-glycans were also synthesized by the same strategy. The synthesized N-glycans were used to probe the molecular basis of H1N1 neuraminidase recognition. The asymmetrically deuterated N-glycans revealed a difference in the recognition of sialic acid on each branch. Meanwhile, the tetraantennary N-glycan was used to evaluate the effects of multivalency and steric hinderance by forming branching structures.

Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid†A.

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.

Characterisation of the Dynamic Interactions between Complex N-Glycans and Human CD22.

CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells.

Weak Agonistic LPS Restores Intestinal Immune Homeostasis.

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c+ cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases.